Micropatterning Proteins

This protocol is based on IgG micropatterning methods as published by Freeman et al. It may need to be optimized for other proteins.

Preparation of the Microprinting Stamp.

This is optimized for a PDMS based stamp and may have to be modified for stamps made of different materials. It is best to perform this procedure after each use of the stamp; the stamp can then be briefly rinsed with 70% ethanol before use.

1. Press the stamp gently against a piece of scotch take, and then pull the tape away.
2. Soak stamp in diluted laboratory glassware cleaner, 20 min.
3. Rinse 5 times with dH₂0.
4. Rinse with 70% ethanol.
5. Allow to air dry.

Patterning a coverslip.

Acid washed &/or plasma cleaned coverslips should be used for this procedure.

1. Dilute IgG to 1 mg/mL, preparing ~20 μL per coverslip to be patterned¹.
2. Tape a piece of parafilm to the bench.
3. Place a 20 μL drop of the IgG solution on the parafilm.
4. Place the stamp, pattern-side down, on top of the drop.
5. Incubate at room temperature for 30 minutes.
6. Lift the stamp and gently dry under a slow flow of air.
7. Place the stamp on a cleaned coverslip and place a light weight (e.g. 1.5 mL tube filled with water) on top of the stamp.
8. After 10-20 seconds, remove the stamp. If preparing other coverslips, repeat steps 3-8 for the additional coverslips.
9. Passivate the coverslip by placing 0.1 mg/mL of poly(L-lysine)-graft-poly(ethylene glycol) [PLL-g-PEG] in PBS for 30 min at room temperature².
10. Rinse once with water and store until needed.
11. Clean the stamp and store, pattern-side-up, in a sealed container.

^1. Unlabeled or fluorescently-labeled IgG can be used. If using unlabeled IgG, a fluorescent secondary antibody can be used to detect the IgG spots after the experiment is completed.

^2. Passivation prevents proteins and cells from adhering directly to the glass. Depending on the substrate, this step may or maynot be needed.